Introduction

Figure 2: Hopanoid lipids are commonly found in outer membranes. The TLC plates showed the lipid distribution of both inner membranes and outer membranes. Both the inner and outer membranes showed the presence of phospholipids. Only the outer membranes showed the distribution of diplopterol and bacteriohopanepolyols.[2]

As major components of the selectively permeable bacterial membranes, lipids, though with relatively monotonic structures compared to membrane proteins, play significant roles in biochemical activities and stress tolerance.[3]Hopanoid lipids are well-studied modern lipid models. They are widely found on a large scale of organisms, such as bacteria, plants, and some lichens, though no hopanoid lipid is found in archaea. Among bacteria, both Gram-negative and Gram-positive bacteria contain hopanoid lipids, which indicates the critical roles they play in bacterial growth and reproduction (Figure 1).[4] Hopanoid lipids are commonly localized in the bacterial outer membranes. Methylobacterium extorquens, one Gram-negative bacterium, mostly produces three types of hopanoid lipids, which includes diplopterol (Figure 3), 2-methyl-diplopterol, and bacteriohopanepolyols (one polar hopanoid lipid).[5] According to the TLC plates, all those hopanoid lipids are only found on the bacterial outer membranes (Figure 2).[2]

Hopanoid lipids are pentacyclic lipids, each of which consists of four six-carbon rings and one five-carbon ring at one end (Figure 4). With the similar structure as the four-ring eukaryotic sterols, hopanoid lipids connect rings via sharing one carbon-carbon single bond between two neighboring ring structures, eventually forming flat, hydrophobic, and stable chemical structures.[6] Hopanoid lipids also contain different hydrophobic and hydrophilic side chains, like R groups in amino acids. As a result, the various side chains increase diversity and expand functions of hopanoid lipids.[7]

The hopanoid lipids play crucial roles in the microbiome and the microbial interaction with animals and plants. From previous studies, the hopanoid lipids, as one of the essential components in a biomembrane, contributes substantially to bacterial membrane permeability and fluidity, stress resistance, nitrogen fixation and bacterial associations with plants, etc.[7] The study of hopanoid lipid, as one of the new and frontier research, remains lots of unknown and interesting topics to study. Although we understand many broad overviews of hopanoid lipids, the detailed mechanisms remain unascertained. Overall, future studies are needed to unveil more interesting facts of hopanoid lipids.[3]

Hopanoid biosynthesis

Hopanoid lipids, with a basic hopane skeleton structure (Figure 4), contain four cyclohexanes and one cyclopentane, all of which connecting each other by sharing one carbon-carbon single bond. Thus, those carbons rings, with the all-chair conformation, form a planar, hydrophobic, and stable structure with thirty carbons.[7] Based on the basic skeleton structure, the hopanoid lipids are further modified by the methylation of the squalene precursor. The most common simple hopanoid compounds in the bacterial membranes are diplopterol (Figure 3) and diploptene (Figure 5). Among the 40 widely-found elongated hopanoids in the bacterial membranes, the most common hopanoids are aminobacterialhexanetriol and bacteriohopanetetrol.[3][7]

Around ten percents of bacteria show the ability to synthesize hopanoid lipids, such as Cyanobacteria and Bacilli.[3] The biosynthesis of hopanoid lipids is generally divided into five different stages. Stage one begins the synthesis of hopanoids through either the mevalonate pathway and the mevalonate-independent pathway. In the mevalonate pathway, bacteria utilize acetyl-CoA, which is produced during bacterial respiration, to synthesize the dimethylallyl pyrophosphate and isopentenyl pyrophosphate.[9] Both products from this mevalonate pathway are used as the five-carbon building blocks. The mevalonate-independent pathway utilizes the glyceraldehyde-3-phosphate and two carbon compounds to produce dimethylallyl pyrophosphate through a series of complicated reactions, such as pyruvate decarboxylation, phosphorylation, and dehydration. The mevalonate-independent pathway, a biosynthesis commonly found in plant plastid organelles, is also found in some wide-known pathogens, such as Mycobacterium tuberculosis.[10] This alternative pathway indicates a potential antibiotic target for bacterial inhibition. Fosmidomycin, isolated from the secondary metabolism of Streptomyces, a genus of Gram-positive soil bacteria, inhibits the activity of 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, one essential enzyme utilized in the mevalonate-independent pathway. Additionally, this enzyme is not produced in the human body, and thus this potential new antibiotic candidate might destroy one of the crucial bacterial enzymes without negatively influencing human body. [7][11][12]

The mechanisms of both stage two and three are similar to that of the sterols synthesis. In stage two, bacteria, with the assist of isopentenyl pyrophosphate, connect two units of dimethylallyl pyrophosphate via head-to-tail condensation, forming one unit of farnesyl diphosphate. In stage three, two units of the product from stage two are reductively connected

through head-to-head condensation with the assist of synthesizing squalene. Most of the final products of stage three show trans conformations with high stability.[7]

In stage four, the squalene proceeds the cyclization reaction to produce hopene. The organic reaction happening in stage four is one of the most complicated one-step organic biosyntheses. The squalene first folds into a proper orientation, therefore increasing the kinetics of this reaction and proceeding the polycyclic formation. This cyclization reaction includes the modification of nine stereocenters, the connection changes of thirteen covalent bonds, and the establishment of four cyclohexanes and one cyclopentane. The discovery of this one-step complicated carbocation formation reaction interested twenty-century organic chemists. In stage five, bacteria modify the unfinished product from stage four through reactions, such as side chain formation and core backbone structure modification. In the side chain formation, various enzymes, especially those coded by the hpn genes, further diversify the structure of hopanoid lipids through adding different types of side chains. The methylation of the core structure happens in the backbone structure modification. A few organisms also conduct the desaturation of the five rings. However, less biochemical mechanisms are known for the stage five, and the details of this process need further studies.[3][7]

Bacterial membrane permeability and fluidity

Figure 6:Hopanoid lipids limit the movement of hydrophobic lipid tails, and therefore increase the stability of the bacterial membrane.[7]

The hopanoid lipids, which contain a major hydrophobic five-ring structure with either polar or non-polar side chains, show mostly hydrophobic and relatively stable characteristics. Thus, the majority of hopanoid lipids can travel and intercalate into the hydrophobic layer of the double membrane and interact with the tails of hydrophobic lipid. In other words, the hydrophobic hopanoid lipids insert between the non-polar fatty acids, decreasing the extra space between the lipid hydrophobic tails. Therefore, due to the decreased movements of the lipid tail, hopanoid lipids increase the membrane thickness and decrease its fluidity (Figure 6).[7] Experimental data also supports this phenomenon. Researchers used three different strains, which includes wild-type M. extorquens (WT), hopanoid-depleted wild-type strain (WT-depleted), hopanoid-deficient shc mutation strain (ΔSHC).

Figure 7:The hopanoid lipids increase the membrane order. The membrane order was detected by the comparison of general polarization values and hopanoid lipids concentration. [2]

They also added diplopterol and cholesterol into the shc mutant. The results showed that the wild-type strain and the mutant with the treatment of diplopterol and cholesterolthe showed higher ordered membrane compared to that of the hopanoid-deleted wild-type and hopanoid-deficient mutation strain. Therefore, the hopanoid lipids increase the membrane order (Figure 7).[2]

Various side chains of hopanoid lipids might have different influences on the bacterial membrane characteristics and properties. For example, the 3-methyl-BHT, with a large hydrophilic side chain, experiences difficulties in inserting into the membrane. However, the diploptene with a hydrophobic side chain relatively freely moves inside the lipid tails. Therefore, hopanoid lipids with unique functional groups affect its distribution inside the bacterial membranes, the way that hopanoid lipids interact with the environment, and interactions with other hopanoid lipids’ function groups. In the membrane dynamic simulation, the amphiphilic hopanoid lipids, with a hydrophobic skeleton and a hydrophilic side chain, show an upright orientation, which is similar to other phospholipids. Due to their unique orientation, the amphiphilic hopanoid lipids can interact with other lipid layers, and thus condense the overall structure and increase the integration of the bacterial membranes. On the other hand, some hydrophobic hopanoid lipids with hydrophobic side chains show a strong ability to transfer into the nonpolar part of the biomembranes, which increases the lipids thickness by limiting the movement of the hydrophobic tails.[3]

The functions of steroid alcohols in eukaryotic and hopanoid lipids in prokaryotes show similarities. Both of them contributes to the membrane stability, but they might contribute to the stability differently. In one dynamic lipid simulation, researchers use cholesterol, one kind of steroid alcohols, and diplopterol, one type of hopanoid lipids, to analyze their ability in membrane organization. Both cholesterol and diplopterol prohibit the formation of gel phase during membrane simulation, and they also order the orientation of N-stearoyl-D-erythro-sphingosylphosphorylcholine, one artificial sphingolipid (Figure 8).[14] However, the cholesterol induces the immiscible formation of both liquid disorder formation and liquid ordered configuration, while the diplopterol shows a separation of those two lipid formations (Figure 8). Thus, those two different configurations of lipids indicate the functional differences of steroid alcohols and hopanoid lipids in the cell membrane. (Figure 9).[14]

Additionally, the influence of either steroid alcohols or hopanoid lipids might be different in vivo. In the artificial membranes, the membranes with the interaction of 2-methyl-diplopterol, one methylated hopanoid lipids, show higher thickness compared that of cholesterol. Thus, the addition of 2-methyl-diplopterol in biomembrane decreases the fluidity and increases the stability. However, the membrane of Rhodopseudomonas palustris strain with a hpnP mutation, one gene that codes for the production of 2-methyl-diplopterol, shows no significant differences in the bacterial membrane thickness compared with that of the wild-type strain with a functional hpnP gene. Therefore, the in vivo condition is more complexed compared to the lipid dynamic simulation.[14]

Stress tolerance

Figure 10:The shc mutation shows sensitivity in high concentration of bile salts condition. However, both wild-type strain and shc complemented strain show resistance to bile salts.[15]

The abundance and diversity of hopanoid lipids positively influence bacterial resistance to environmental stress, such as the extreme pH, high pressure, non-optical temperature, and high concentration of antibiotic or other lethal chemical compounds.[3] With the help of hopanoid lipids, microbiomes develop different strategies to increase the fitness in non-optical environments. However, most of the detailed mechanisms of stress tolerance induced by the hopanoid lipids remain unclear and require further studies.[3]

Methylation of hopanoid lipids

Researchers found that under extreme conditions, the biosynthesis of hopanoid lipids increases in most bacterial strains. As a result, hopanoid lipids increase bacterial stress resistance.[3] For example, When Rhodopseudomonas palustris is placed under extreme environments, such as high temperature or low pH conditions, those stress induces the activation of the ecfG gene, which is a general stress regulator for the alphaproteobacteria. The response factors and regulators synthesized by ecfG upregulate the expression of hpnP, a gene that codes for hopanoid methylases. Therefore, the expression of hpnP increases the rate of hopanoid lipids methylation in bacteria. Methylation increases the hydrophobic characteristics of hopanoid lipids and further stabilizes the movement of phospholipids hydrophobic tails, perhaps increasing the bacterial membrane resistance to potential dangers [16].

Membrane fluidity and permeability

The presence of hopanoid lipids decreases the fluidity and permeability of bacterial membranes. The hydrophobic part of the hopanoid lipids migrates into the phospholipid tails, and the hydrophilic side chains also form stronger attractions with other molecules. Thus, hopanoid lipids fill the empty spaces and increasing the membrane integrity. Overall, the biomembrane becomes more stable, and the leakage of lethal molecules decrease. Under the low pH or high antibiotic condition, the proton and antibiotic compounds might experience difficulty across the bacterial membranes due to decreased permeability. Therefore, bacteria show higher resistance towards the high concentration of proton, antibiotics, and other lethal molecules.[3]

Different hopanoid lipids show different contributions towards membrane fluidity and permeability. Bile salts show no negative influence on majority of the Gram-negative bacteria because these chemical compounds are not able to transfer across the outer membrane of the Gram-negative bacteria. Mutated bacteria, without the protection of hopanoid lipids, are vulnerable to the high bile salt conditions. However, different mutations in the same bacteria species show different levels of bile salts sensitivities. shc gene codes for the production of diplotene and diplopterol. The Rhodopseudomonas palustris with this mutation show no bile salts tolerance (Figure 10).[15] The hpnH mutation causes the deficient production of diplotene and diplopterol. Rhodopseudomonas palustris with this mutation show resistance towards the bile salts but perform a slower growth rate compared to that of the wild-type strain. The hpnO mutant cannot produce aminobacteriohopanetriol, one essential bacterial hopanoid lipid. This mutant shows no influence on bacterial growth and culture density in the high bile salts concentration compared to the wild-type strain.[3][15]

Orientation and distribution of hopanoid lipids

The orientation and the distribution of hopanoid lipids contribute to the bacterial heat resistance. Bacteria, with the help of high concentration of hopanoid lipids, obtains heat protection and keep the homeostasis of their cytoplasm. As the environmental temperature increases, the hopanoid lipids tend to move to the place between two leaflets. In the lipid dynamic simulation at room temperature (298 K), most of the diplopterol molecules gather in the area between the head and the tail of phospholipid. Thus, at milder temperature, the distribution of hopanoid lipids shows a large separation. However, as the temperature increases, the distribution of diplopterol molecules migrate to the empty spaces between the tails of two leaflets, largely increasing the integration and thickness of the bacterial membrane, creating a substantial protection for the thermophiles (Figure 11).[17]

Figure 13: The intracellular concentration of NPN of the shc mutant strain does not change a lot, while that of the wild-type strain decreases constantly.[2]

Interaction with bacterial membrane proteins

The Methylobacterium extorquens mutant strain, with a non-functional hopanoid lipid production, shows an increased detergent sensitivity (Figure 12). Additionally, this strain perhaps cannot pump out the antibiotics due to its non-functional efflux. Researchers studied the accumulation of 1-N-phenylnaphthylamine (NPN), one type of lipophilic dye. The wild-type strain constantly pumps out NPN, and therefore the intracellular concentration of PNP keeps decrease. However, the intracellular concentration of shc mutant does not change a lot, indicating that perhaps the mutant has deficient efflux (Figure 13).[2] Thus, the hopanoid lipids might contribute and assist the function of membrane proteins.[3] The collaboration of the hopanoid lipids and membrane proteins enhance bacterial resistance and help bacteria to maintain the homeostasis between the cytoplasm and extracellular environment. Additionally, bacterial membranes, one of the most significant energy production places, requires the presence of effective hopanoid lipids to achieve energy production and storage. Nostoc punctiforme, with a hopanoid lipid mutation, show a decrease in energy storage compared to that of the wild-type strain.[3]

The function of hopanoid is more significant in the stress-resistance specified cell. The filament ofNostoc punctiforme, one kind of filamentous cyanobacteria, are constituted by the vegetative cells. That specified cells conduct photosynthesis under the environment with abundant nutrients. Therefore, they fix carbon dioxide in the environment and show a high growth and reproduction rate. In these photosynthetic cells, the hopanoid concentration shows negligible influence on the stress response. However, under the environment with poor nutrient concentration, Nostoc punctiforme develop the akinete cells through differentiation, which contain higher resistance toward the cold and dry environment. The akinete cells keep their stable structure and bacterial activity. They also stay in low energy consumption and store energy for many years until they encounter an environment with rich resources. In the akinete cells, a high concentration of hopanoid lipids is detected in their bacterial membranes. Previous studies found that the mutation without functional hopanoid lipid shows less resistance to the extracellular resistance. Similarly, Streptomyces coelicolor, one Gram-positive soil bacteria species, forms vegetative cells under nutrient-rich environment. These vegetative cells have a low hopanoid lipid concentration. However, at the end of their life cycle, they produce and accumulate hopanoid lipids and form spore via sporulation, which can assist their resistance under the extreme condition[3].

Nitrogen fixation

Figure 14: The nodules formed by wild-tryp, hpnP, and hpnH strains. Compared to the wild-type B. diazoefficiens, the nodules formed by either hpnP or hpnH strain are unhealthy. CDC.

Various bacteria, with the ability to produce hopanoid, can fix nitrogen. Bacteria, such as Beijerinckia, Frankia, Anabaena, Burkholderia, etc., show a positive correlation between the hopanoid production and nitrogen fixation.[3] Most nitrogen-fixing bacteria live in a close physical correlation with plants, such as alfalfa, soil beans, peas, etc. Both bacteria and plants can take advantage of this relationship. Bradyrhizobium spp. derive energy and other important chemical cofactors from their host plants. The plants also can protect them from the stress in the environments, such as competitions with other microbiomes, high proton concentration, etc. Equally, plants benefit from the nitrogen oxides produced by the nitrogen-fixing bacteria, and therefore plants show an enhanced growth and outcompete their competitors. There are also free-living nitrogen-fixing microbes, such as Anabaenaspp.,Frankia spp., etc. However, most of those bacteria do not fix nitrogen without coexisting with plants.

The legume-rhizobia root nodule symbiosis is among one of the most widely studied plants and bacteria associations. The production of hopanoid lipids influences the bacterial symbiosis with the host plants. TheBradyrhizobium spp., with shc mutation, produce negligible amount of hopanoid lipids. Therefore, this bacteria strain is incapable of integrating with plants. Additionally, different types of hopanoid lipid contribute differently to the bacterial association with plants. The Bradyrhizobium diazoefficiens with a hpnP mutation cannot produce the extended hopanoids. Their association with the Aeschynomene afraspera results in a morphologically disorganized nodule. The Aeschynomene afraspera show nitrogen starvation, indicating a decreased nitrogen-fixing ability of the hpnP mutant. At the same time, the Bradyrhizobium, with a hpnP mutation, show no production of 2-mehopanoids. The nitrogen fixation of this mutation strain, in contrast, show no differences compared to that of the wild-type strain (Figure 14)[18].

The presence of the hopanoid lipid might also contribute to the competition between bacteria to inhabit the hosts. The mutation strain shc, which produces no hopanoid lipids shows an inability in long-term colonization of a plant. Previous studies have found that in the beginning, these bacteria grow and develop in host normally. However, after a few days, these bacteria starts to degenerate and be recycled by their host. For example, the Bradyrhizobium and Burkholderia, both of which contain a high concentration of hopanoid lipids, show high resistance to high temperature and low pH conditions. Thus, in consideration of global warming and increasing soil acidity, both of those bacteria might outcompete other bacteria in soil and plant.

Figure 15: Under the high concentration of oxygen, Azotobacter vinelandii also synthesize alginate to create alginate capsule to protect nitrogenase activity. CDC.

Bacteria conduct the majority of nitrogen fixation in the low-oxygen concentration environment. Nitrogenase, the core enzyme in nitrogen fixation, is sensitive and vulnerable in the presence of oxygen. Thus, the majority of the nitrogen-fixing bacteria exist under the anaerobic environment. Sometimes, nitrogen fixation can also be conducted at high oxygen concentration. Under this condition, bacteria develop more sophisticated mechanisms to protect the nitrogenase and achieve nitrogen fixation. The free-living nitrogen-fixing bacteria, Azotobacter vinelandii, develop various ways to fix nitrogen under high oxygen condition. Previous studies observed that the activity of nitrogenase activity has negligible decrease under the air saturation from 30-100%. The Azotobacter vinelandiidecrease their cellular surface area. Therefore, for each unit of cytoplasm, there is larger protection from the bacterial membrane. Also, under high oxygen condition, they also increase their oxygen consumption via a high respiration rate, thus creating an optional anaerobic environment for nitrogen fixation. The Azotobacter vinelandii also synthesize alginate to create alginate capsule, a thick barrier which limits the oxygen fission and protects the nitrogenase from the high oxygen-concentrated environment. Under the lower oxygen concentrated environment, Azotobacter vinelandiidevelops a loose alginate capsule, while under the high concentration, they build up a compact and thick alginate capsule (Figure 15). Additionally, when the bacteria interacts with or inhabit plants, the host plants synthesize leghemoglobin molecules, one type of oxygen-transport metalloproteins. This hemoglobin scavenges oxygen from the root nodules of the leguminous plants, creating an optimal environment for nitrogenase activity[19].